PROJECT SUMMARY There is a growing appreciation that diet-gut microbiota interactions play an important role in human cardiovascular health. Recent studies have discovered a diet-derived, gut microbiota-dependent metabolite, trimethylamine N-oxide (TMAO), which may promote the development of cardiovascular disease (CVD). So far, only a few small studies among general populations have investigated this novel TMAO-CVD pathway. Limited evidence suggests that TMAO production and metabolism is influenced by both dietary and non-dietary factors, and that the diet-TMAO association may vary across populations due to significant variations in TMAO food sources (e.g., meat vs. fish vs. plant foods). Large-scale, collaborative epidemiologic studies that include ethnically and regionally diverse populations and incorporate comprehensive data on diet, TMAO metabolites, and CVD biomarkers will provide critical insights into this meta-organismal pathway and may pave the way for developing new strategies for CVD prevention and treatment based on diet-gut microbiota interactions. We propose to conduct a large, international, collaborative study: the TMAO Pooling Project. It will be primarily based on the newly established Consortium of Metabolomics Studies (COMETS) and will also include several other population-based studies. As of Oct. 2017, we have recruited 18 studies from the US, Europe, and Asia, bringing a total of 44,164 participants, including 51% women and 46% understudied racial/ethnic groups (i.e., blacks, Hispanic/Latinos, and Asians). Our Specific Aims are: 1) to examine distributions of TMAO and related metabolites (i.e., choline, carnitine, and betaine) across populations by age, sex, region, race/ethnicity, obesity, smoking status, and metabolic disease status; 2) to evaluate associations of TMAO metabolites with intakes of trimethylamine-containing foods including potential variations across populations; and 3) to evaluate associations of TMAO metabolites with known CVD biomarkers (e.g., glucose, blood lipids, and inflammatory markers). We hypothesize that TMAO level may differ across populations due to variations in habitual diets and metabolic disease status, which in turn may affect CVD health in these populations. This will be the first, large-scale, multi-ethnic, multi-national metabolomics study that leverages existing data and consortium resources to investigate this novel diet-TMAO-CVD pathway. It is feasible and extremely time- and cost-efficient. Knowledge gained will help elucidate the distribution, influencing factors, and potential effects of TMAO on CVD. This study will lay a foundation for future studies on the role of diet-gut microbiota interactions in the development of CVD among diverse populations, which may eventually lead to new, population-specific strategies for CVD prevention and treatment via dietary and/or gut microbial modifications. Meanwhile, this study will establish a network for collaborations on emerging topics in CVD research such as gut microbiota, metabolomics, and health disparities, and will foster career development of junior investigators in these areas as they collaborate and learn from senior investigators and their peers.